Inhibiting or preventing thrombus formation with 2-methylthioadenosine-5{40 -monophosphate

ABSTRACT

2-Methylthioadenosine-5&#39;&#39;-monophosphate and the ammonium, sodium and potassium salts thereof and pharmaceutical compositions and methods of inhibiting or preventing thrombus formation with these compounds.

United States Patent Maguire et a].

[is] 3,678,162 1451 July 18, 1972 [54] INHIBITING OR PREVENTING THROMBUSFORMATION WITH 2- METHYLTHIOADENOSINE-S MONOPHOSPHATE I [72] Inventors:Mary H. Maguire; Geoffrey R. Cough,

both of Mosman; Frank Michal, Lane Cove, New South Wales, all ofAustralia [73] Assignee: The University of Sydney, Sydney, New

South Wales, Australia [22] Filed: Dec. 8, 1969 211 App]. No.: 883,354

[30] Foreign Application Priority Data Dec. 13, 1968 Australia ..4778952 us. c1. ..424/180, 260/211.5 R 511 1111.01. ..A6lk 27/00 PrimaryExaminer-Jerome D. Goldberg Assistant Examiner-Vincent D. TurnerAttorney-William H. Edgerton, Richard D. Foggio, Joan S. Keps, Alan D.bourie and Joseph A. Marlino [57] ABSTRACTZ-Methylthioadenosine-S'-monophosphate and the ammonium, sodium andpotassium salts thereof and pharmaceutical compositions and methods ofinhibiting or preventing thrombus formation with these compounds.

4 Claims, No Drawings INHIBITING OR PREVENTING THROMBUS FORMATION WITHZ-IVIE'HIYL'I'THOADHQOSINE-S MONOPHOSPHATE This invention relates to newchemical compounds, namely 2-methylthioadenosine-5'-monophosphate andthe ammonium, sodium and potassium salts thereof. This invention alsorelates to pharmaceutical compositions and methods of in hibiting orpreventing thrombus formation with these compounds.

lt has been found that 2-methylthio-adenosine-5'- monophosphate isuseful for the inhibition or prevention of thrombus fonnation in, forexample, post-operative situations, cases of heart implants withpacemakers rnd artificial valves, organ transplants, atherosclerosis,venousthrombotic states and conditions where platelet adhesion isexcessive.

The activity of Z-methylthioadenosine-S-monophosphate is demonstrated bythe following procedures.

Z-Methylthioadenosine-S'-monophosphate at 0.5 p.M causes a 50 percentinhibition of adenosine diphosphate (ADP)-induced aggregation of sheepblood platelets in platelet-rich plasma in vitro. Similarly a 50 percentinhibition of the ADP-mediated aggregation of dog and human platelets iscaused by 1.3 M and 8.6 M 2-methylthioadenosine-5'- monophosphate. Theinhibition produced is of long duration.

The technique used for the quantitative measurement of plateletaggregation is described in the article by M.l-l. Maquire and F. Michal,Nature, 217,571 (1968). 2- Methylthioadenosine-S-monophosphate reducesthe stickiness of platelets in vivo, demonstrated by the followingprocedures.

Z-Methylthioadenosine-S'-phosphate (250-500 rig/kg) was administered todogs anaesthetized with nembutal or chloralose and readministered atintervals of 60 to 90 minutes. Blood was sampled at hourly intervals,and the response to ADP of the platelets in plateletrich plasma isolatedfrom the blood samples was studied. A significant reduction in theADP-induced aggregation of platelets was observed, and the plateletaggregates formed disaggregated more rapidly than normal. Results wereobtained from experiments on seven dogs.

Thrombi can be induced in exposed cerebral cortex ar- 5-monophosphatedoes not produce arrhythrnias or heartblock and does not causesubstantial depression of arterial blood pressure.

Doses of 75 mg/kilograrn administered intravenously in mice showed notoxic effects.

Z-Methylthioadenosine-S'-monophosphate may be prepared for example from2-methylthioadenosine by either According to the above procedure, the 2'and 3'-hydroxy groups of 2-methyl-thioadenosine are protected bytreating with acetone in the presence of an acid condensing agent suchas for example p-toluene sulphonic acid to give 2-methylthio-2',3-isopropylideneadenosine.

The 2-methylthio-2,3'-isopropyl-ideneadenosine is then reacted withB-cyanoethylphosphate in a solvent such as pyridine, usingdicyclohexylcarbodimide as a condensing 0 acid -monophosphate. Thisproduct may be purified by treating agent.

The B-cyanoethyl and isopropylidene groups are removed with,respectively, alkali (for example lithium hydroxide) and (low pH) to.give 2-methyl-thioadenosine-5- with barium hydroxide to give the bariumsalt, converting the barium salt to the ammonium salt by ion-exchangechromatograph purifying the ammonium salt by column chromatography andthen converting to the free acid, 2-methylthioadenosine-S'-monophosphate, by elution from an anion exchangecolumn with formic acid.

Alternatively the 2-methylthio-2,3'-isopropylideneadenosine is treatedwith phosphorus oxychloride in the presence of trimethyl or triethylphosphate. Excess phosphorus oxychloride is hydrolyzed with aqueouslithium hydroxide. Lithium phosphate is removed the solution is heatedat pH 2 for 30 minutes and 24nethylthioadenosine-S'- monophosphate isisolated as the barium salt, and purified as described in the firstprocedure.

The free acid may be convened to its sodium or potassium salt by passagethrough a sodium or potassium cation exchange column and evaporation todryness.

For therapeutic use, 2-methylthio-adenosine-5'- monophosphate ispreferably administered in the form of a pharmaceutical compositioncomprising the active ingredient in combination with a pharmaceuticalcarrier. These pharmaceutical compositions also fall within the scope ofthe present invention. Advantageously, the compositions can be made upin a dosage unit form appropriate to the desired mode of administration,which may be intravenous or intramuscular.

The pharmaceutical carrier may be for example, pyrogen free normalsaline.

The following examples illustrate the invention and are not intended tolimit the scope thereof.

EXAMPLE 1 2-methylthio-2',3 O-isopropylidene adenosine a. PureZ-methylthioadenosine (2 g) and p-toluene sulphonic acid (12.8 g) weresuspended in dry acetone (450 ml) and the mixture was stirred for 90minutes with a Vibromix. After 10 min. a clear solution was obtained,after 30 min. crystals separated. The mixture was poured into 350 ml ofice water containing 13.4 g of NaHCO and the solution obtained wasevaporated to dryness at 30-35.

The residue was extracted by repeated heating of the reaction productwith acetone (8 X 500 ml) and decanting of the acetone extract.Evaporation of acetone left an oil which was dissolved in 500 ml ofboiling H O, charcoaled and the solution evaporated to 20 ml in vacuo at30. Crystals separated and were filtered (1.55 g) and crystallized againby evaporation from an aqueous methanolic solution. The aqueous motherliquors yielded another 200 mg and recrystallization of the combinedproducts from H O gave needles (1.4 g) mp 177-l78.5, chromatographicallyhomogeneous. Analysis: C H NO S req. C 47.6; H 5.38; N 19.8. Found:47,66; 1-1 5.28; N 19.70%.

It has been found advantageous to use a dimethoxypropane acetone (1:10)mixture in the initial acetonation step, to use a longer reaction time,and to extract the product from the NaHCO evaporation residue withchloroform instead of acetone.

b. 2-Methylthioadenosine (2.5 g) and p-toluene sulphonic acid (16 g)were suspended in dry acetone (750 ml) containing 2,2-dimethoxypropane(40 ml) and the mixture was stirred for 4 hours, then poured into icewater (400 ml) containing NaHCO (18.2 g). The solution was evaporated todryness and the residue was dried in vacuo over NaOH. The residue wasthen extracted with boiling chloroform (3 X 100 ml). Evaporation ofchloroform left an oil which was crystallized from hot water yielding2.2 g (78 percent of theory) of the pure product.2-Methylthioadenosine-5-monophosphoric acid a. 1.06 g (3 moles) of2-methylthio2,3-O-isopropylidene adenosine in 25 ml pyridine was allowedto stand at room temperature with 12 ml of l mmoles/ml stock solution ofB- cyanoethyl phosphate and 6 g of dicyclohexylcarbodiimide. Thereaction was terminated after 24 hr. by the addition of ml of water andthe resultant mixture evaporated to dryness. 120 ml of 0.4N lithiumhydroxide solution was added to the residue and the mixture was refluxedfor 1 hr to remove the cyanoethyl protecting group. It was then cooledand filtered, and the filtrate was passed through a column of Bio-Rad AG50W-X4 cation exchange resin (l-l form) to replace lithium by hydrogenions. The acidic effluent was left at room temperature overnight toeffect cleavage of the isopropylidene protecting group. It was thenconcentrated to about 150 ml and its pH adjusted to 7.5 with bariumhydroxide solution. Barium phosphate which precipitated was centrifugedoff and the supernatant was treated with two volumes of ethanol andallowed to stand overnight. The crude nucleotide barium salt which thensettled out was collected by centrifugation, washed with ethanol,acetone and ether and dried, yielding 1.9 g.

300 mg of crude barium salt was converted to the ammonium salt bypassage through a column of cation exchange resin. It was then appliedin a small volume of isopropanol: 0.25 M aqueous ammonium bicarbonate(2:1) to a 2.5 X 35 cm column of cellulose packed in the same solvent.Elution with this solvent yielded a pure fraction of the desirednucleotide as the ammonium salt. Ammonium bicarbonate from the solventwas removed by repeated evaporations of water and the residue wasconverted from the ammonium salt form to the free acid by absorbing iton a column of Bio-Rad AG1-X4 anion exchange resin, washing the columnwell with water, then eluting it with 4N formic acid. The fractions ofthe effluent which contained the nucleotide were concentrated and driedto yield 2-methyl-thioadenosine-5monophosphoric acid as a white solidmg, approx. 64 percent yield from the iso-propylidene derivative).

The solid was recrystallized twice from water to give white needles, mp.192-l95 C (decomposition). Analysis: C H NO PS. H O req. C,32.12;1-l,4.41; N,17.03; P,7.53. Found: C,3l.77; 1-1,4.42; N,16.94; P, 7.45percent.

The isolation of Z-methylthioadenosine-S'-phosphate may also be achievedadvantageously without proceeding via the barium salt as describedhereunder in (b).

b. 1.06 g of 2-methylthio-2',3'-O-isopropylidene adenosine was treatedas described above up to the step involving reflux with 0.4N lithiumhydroxide. The reaction mixture was then filtered and the filtrate wasadjusted to pH 1.5 with 89 percent phosphoric acid. The acid solutionwas heated at 70-80 C for 2 hrs, then treated with dilute lithiumhydroxide solution until the pH reaches 9. Precipitated lithiumphosphate was filtered off. The filtrate was evaporated to c. 30 ml. andpassed through 2.0 X 30 cm column of Bio-Rad AG 50W X4 (11*) whichretained the nucleotide. The column was washed with 500 ml water theneluted with 300 ml of l M Nl-LOH. The eluate was evaporated to 15 ml.then strongly acidified with formic acid.2-Methylthioadenosine-5-monophosphate began to crystallize outimmediately.

The mixture was refrigerated overnight then filtered to give 899 mg ofchromatographically homogeneous white needles (73 percent yield).

A further 266 mg of crude product was isolated from the mother liquor byabsorption on a column of Bio Rad AG2X8 and elution with 4M fomlic acid.This was recrystallized once from water to give the pure product (197mg, total yield 89 percent).

c. 1.06 g (3 mmoles) 2 methylthio-2, 3'-O-isopropylidene adenosine wasdissolved in a solution of 2.73 ml (30 mmoles) phosphoryl chloride and1.43 ml (6 mmoles) of tri-n-butylamine in 5 ml dry trimethyl phosphateat 0 C. After 72 hrs at 0 C, the reaction mixture was poured into water(250 ml), the pH adjusted to 1.5 with LiOH solution, and the resultingsticky mass stirred at 70 C for 1 hr during which time most of itdissolved. The solution was adjusted to pH 9 with LiOl-l and theprecipitate of lithium phosphate filtered off. The filtrate was passedthrough a 2.5 X 35 cm column of Bio Rad AGSOW-X4 (H') which retained thenucleotide. The column was washed with 300 ml water then eluted with 1MNH OH. The alkaline eluate was evaporated to c. 50 ml then applied to a3.2 X 28 cm column of Bio-Rad AG2X8 (formate form) and followed by 500ml water. Elution was then begun with 4M HCOOl-l. 15 ml fractions werecollected. 2- Methylthioadenosine-S-monophosphate emerged in fraction34-43. These fractions were pooled and incorporated to give 260.3 mgwhite crystalline product (21 percent yield).

EXAMPLE 2 Z-Methylthioadenosine-S'-monophosphate in water is passedthrough a sodium cation exchange resin and the solvent is removed byevaporation in vacuo to give the sodium salt of2-methylthio-adenosine-5monophosphate.

prising a pharmaceutical carrier and an efiective thrombus inhibiting orthrombus preventing amount of 2- methylthioadenosine-S'-monophosphate.

3. A method of inhibiting or preventing thrombus fonnation whichcomprises administering internally to an animal an effective thrombusinhibiting or thrombus preventing amount of Z-methylthioadenosine-S-monophosphate or the ammonium sodium or potassium salt thereof.

4. A method according to claim 3 in which 2-methylthioadenosine-S'-monophosphate is administered.

s1 WARD M.FL'ETCI-HER,JR.

(5/69) UNITED STATES" PATENT. OFFICE CERTIFICATE OF CORRECTION PatentNo; 3,618,162. Dated 1 18 1972 w Inventor-(s) 'r Ma uire Geoffre R. on hand Frank .Michal It is certified that error appears in theabove-identified patent and that said Letters Patent are herebycorrected as shown below:

f'olumn 1, lines 62 to 75, deletethe right-hand formula and in? line 65delete the arrow following the right-hand formula. Column 2, lines l to24, should appear as follows:

Column 2, lines 31 to 33, in the left-hand formula,

that portion of the formula reading uo-i -o-cn should read" H0-l O-CH OHOH a Column 5, line 10, "-monophosphate" should read2-=-methylkhioadenosine-S'-monophosphate.

Signed and sealed this 26th day of December 1972.

(:sEAL) A test:

- ROBERT GOTTSCHAELK A testing Officer Commissioner of Patents

2. A pharmaceutical composition according to claim 1 comprising apharmaceutical carrier and an effective thrombus inhibiting or thrombuspreventing amount of 2-methylthioadenosine-5''-monophosphate.
 3. Amethod of inhibiting or preventing thrombus formation which comprisesadministering internally to an animal an effective thrombus inhibitingor thrombus preventing amount of 2-methylthioadenosine-5''-monophosphateor the ammonium, sodium or potassium salt thereof.
 4. A method accordingto claim 3 in which 2-methylthioadenosine-5''-monophosphate isadministered.